Co., St. Louis, MO, U.S.A. Endoglycosidase H was from Miles. Scientific, Miles ..... glycoproteins (Hubbard & Ivatt, 1981; Dunphy & Rothman,. 1985). Although we ...
Biochem. J. (1990) 267, 697-702 (Printed in Great Britain)
697
Purification and characterization of the invertase from Schizosaccharomyces pombe A comparative analysis with the invertase from Saccharomyces cerevisiae Sergio
MORENO,*j Yolanda SANCHEZ* and Luis RODRIGUEZt
*Departamento de Microbiologia, Genetica, Medicina Preventiva y Salud Puiblica, Facultad de Biologia, Universidad de Salamanca, 37007 Salamanca, and tDepartamento de Microbiologia y Biologia Celular, Facultad de Farmacia, Universidad de La Laguna, 38071 La Laguna, Tenerife, Spain
Invertase (EC 3.2.1.26) was purified to homogeneity from exponentially growing cells of Schizosaccharomyces pombe fully de-repressed for synthesis of the enzyme, and was shown to be a high-molecular-mass glycoprotein that can be dissociated in the presence of 8 M-urea/l % SDS into identical subunits with an apparent molecular mass of 205 kDa. The carbohydrate moiety, accounting for 67 % of the total mass, is composed of equimolar amounts of mannose and galactose. There is a small amount of glucosamine, which is probably involved in the linkage to the protein moiety, since the enzyme is sensitive to treatment with endoglycosidase H. The composition of the carbohydrate moiety resembles that found in higher-eukaryotic glycoproteins and differs from glycoproteins found in Saccharomyces cerevisiae. The protein portion of each subunit is a polypeptide of molecular mass 60 kDa, very similar to the invertase of Sacch. cerevisiae. Both proteins cross-react with antibodies raised against the protein fractions of the other, indicating that the two enzymes are similar.
INTRODUCTION The fission yeast Schizosaccharomyces pombe, like Saccharomyces cerevisiae, synthesizes invertase that is secreted into the cell wall. In both yeasts biosynthesis of the enzyme is regulated by catabolite repression (Gascon & Ottolenghi, 1967; Mitchison & Creanor, 1969), and, when exponentially growing cells are transfered to a medium with a low percentage of glucose, enzyme synthesis is de-repressed. Sacch. cerevisiae cells contain two forms of the enzyme: one located intracellularly without carbohydrate, which corresponds to the repressed form of invertase, and a second extracellular one, containing nine or ten Nglycosidically linked oligosaccharides, which corresponds to the de-repressed form of the enzyme (Neuman & Lampen, 1967; Gascon & Lampen, 1968; Gascon et al., 1968; Tarentino et al., 1974; Huffaker & Robbins, 1983). Both enzymes are synthesized from the same structural gene, and their protein moieties have a molecular mass of 60 kDa (Carlson & Botstein, 1982). In the fission yeast invertase is a high-molecular-mass glycoprotein located outside the plasma membrane (Moreno et al., 1985). Since study of the Sacch. cerevisiae invertase has yielded valuable information on the nature of yeast glycoproteins (Cohen & Ballou, 1981), we have purified and characterized the invertase from Schizosacch. pombe both in order to compare it with the similar enzyme in Sacch. cerevisiae and to gain information about the composition of Schizosacch. pombe glycoproteins. MATERIALS AND METHODS
Materials
Reagents for gel electrophoresis and Bio-Gel A-Sm were obtained from Bio-Rad Laboratories, Watford, Herts., U.K. DEAE-Sephadex A-50 and ampholytes for isoelectric focusing (Pharmalyte 2.5-5) were from Pharmacia-LKB, Uppsala, Sweden. Proteins used as molecular-mass markers, Sacch. cerevisiae invertase, 2,3,5-triphenyltetrazolium chloride and
Staphylococcus aureus proteinase V8 were from Sigma Chemical Co., St. Louis, MO, U.S.A. Endoglycosidase H was from Miles Scientific, Miles Martin, Madrid, Spain. [35S]Methionine and nuclease-treated reticulocyte lysate were from Amersham International, Amersham, Bucks., U.K. Oligo(dT)-cellulose was from Collaborative Research, Bedford, MA, U.S.A. Inactivated Staph. aureus cells (IgG Sorb) were from The Enzyme Center, Malden, MA, U.S.A. All other reagents were of analytical grade. Enzyme purification Sacch. cerevisiae invertase was obtained from Sigma Chemical Co. and then purified by gel filtration on a Sephacryl S-300 column (1.25 cm x 100 cm) equilibrated in 50 mM-Tris/HCI buffer, pH 7.5. For production of Schizosacch. pombe invertase, the wild-type strain Schizosacch. pombe 972 h- was grown in a 14-litre New Brunswick glass fermenter in yeast-extract medium containing 3 % (w/v) glucose; 4 litres of medium were grown overnight at 30 °C, with shaking at 200 rev./min and aeration at 10 litres/min, to a density of 2 x 107 cells/ml. Then the culture was diluted with 8 litres of fresh medium containing 0.25 % glucose, and incubation was continued for a further 15 h under the same conditions. The cells were harvested and washed twice in ice-cold distilled water and once more with 0.1 M-sodium acetate buffer, pH 4.0. With this procedure it was possible to obtain about 100 g wet wt. of cells in which invertase synthesis was fully de-repressed. Cells were resuspended in 100 ml of 0.1 Msodium acetate buffer, pH 4.0, and broken by mechanical shaking with an equal volume of acid-washed glass beads (0.45 mm diam.) at 4 °C in a Braun MSK homogenizer. Unless otherwise noted, all further procedures were carried out at 4 'C. The extract was spun first at 5000 g for 5 min and then at 40000 g for 30 min. The pH of the supernatant of the second centrifugation was adjusted to 4.0 with 1.0 M-acetic acid, and the solution was left overnight in an ice bath and then centrifuged at 40000 g for 30 min. Solid (NH4)2SO4 was added slowly to the supernatant to 85 % satu-
t To whom correspondence should be sent, at present address: Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OXI 3QU, U.K. Vol. 267
698 ration (56 g/l). After standing overnight in the cold, precipitated material was removed by centrifugation at 40000 g for 30 min. The supernatant, which contained all the enzymic activity, was first dialysed against distilled water and then against 0.1 Msodium acetate buffer, pH 4.0. Then 6 g of DEAE-Sephadex previously equilibrated in the same buffer was added to the resulting solution. The gel slurry was stirred for 2 h and then packed into a column and washed with 2 litres of buffer containing 10 mM-NaCl, after which the enzyme was eluted with 1 litre of buffer containing 50 mM-NaCl. The enzyme solution was concentrated in an Amicon ultrafiltration cell fitted with an XM-100 membrane and then applied to a DEAE-Sephadex column (2 cm x 15 cm) equilibrated in 0.1 M-sodium acetate buffer, pH 4.0, containing 10 mM-NaCl. The column was washed with 500 ml of this buffer and then eluted with 600 ml of a linear gradient of 10-100 mM-NaCl in the same buffer. Fractions with enzymic activity were pooled, dialysed against 0.1 M-sodium acetate buffer, pH 4.0, concentrated to 4 ml by ultrafiltration and filtered through a Bio-Gel A-5m column (2.5 cm x 175 cm) equilibrated in the same buffer. Fractions with activity were pooled, concentrated and rechromatographed in the same way. SDS/PAGE of this material revealed a single protein band. The purified invertase was desalted by dialysis against distilled water, freeze-dried and stored at -20 °C; these conditions proved to be adequate for preserving the enzyme over long periods (over 6 months) without any appreciable loss of activity. Invertase activity Invertase activity was measured by the method of Goldstein & Lampen (1975) as described previously (Moreno et al., 1985). One unit of invertase is the amount of enzyme that hydrolyses sucrose to yield 1 ,umol of glucose/min at 30 °C and pH 4.0.
Protein concentration and amino acid analysis Protein concentration was measured by the method of Lowry et al. (1951), with BSA as standard. Samples for amino acid analysis were hydrolysed in constant-boiling HCI at 110 °C for 24 and 48 h in sealed evacuated tubes. Analyses were performed in a Beckman 119 autoanalyser, fitted with 6 mm (internal diam.) columns, by the procedure described in the Beckman Aminoacid Analysis Manual.
Carbohydrate analysis Total carbohydrate was measured by the phenol/H2SO4 method (Dubois et al., 1956), with mannose as standard. Individual sugars were identified by g.l.c. of their alditol acetate derivates as described by Albersheim et al. (1967) in a Varian 2400 chromatograph. Samples for sugar analysis, containing inositol (1 mg/ml) as internal standard, were previously hydrolysed in 2 M-trifluoroacetic acid at 121 °C for 1 h in sealed evacuated tubes. Identification of neutral sugars was performed on a 3 % ECNSS-M column at 190 °C and amino sugars were separated on a 3 % SP2340 column at 180 °C for 6 min, after which the temperature was increased by 2 °C/min to 300 °C; in both cases injector and detector temperatures were 210 °C and 250 °C respectively, and the carrier gas was He at 60 ml/min. Electrophoresis SDS/PAGE was performed by the method of Laemmli (1970); in some experiments, to avoid denaturing conditions for the samples, SDS was omitted from both the gel and the buffers. Linear gradient (5-15 %) SDS/PAGE was carried out as described by Hames (1981), and two-dimensional electrophoresis was performed as described by Pollard (1984), except that the treatment of the samples with DNAase and RNAase was omitted.
S. Moreno, Y. Sanchez and L. Rodriguez
Proteins were detected either by staining the gels with Coomasie Brilliant Blue R-250 by the procedure of Hames (1981) or by the AgNO3 method of Morrisey (1981). Invertase activity was detected in situ in the gels by the method of Gabriel & Wang (1969) as modified by Carlson et al. (1981). Isoelectric focusing was performed on a 3 %-polyacrylamide/0.6 %-agarose vertical gel slab as described by An der Lan & Chrambach (1981). Antibody preparation A 1 mg portion of purified enzyme was denatured by boiling in 0.1 0% SDS/100 mM-2-mercaptoethanol, and then digested with endoglycosidase H by the procedure of Tarentino et al. (1974). Protein fractions were purified by SDS/PAGE, and the 60 kDa bands were cut out and electro-eluted in 25 mM-Tris/ glycine buffer, pH 8.4, at 100 V for 2 h. Samples containing 100 jug of pure protein were injected into rabbits every 28 days. After the fourth injection the rabbits were bled out and the serum was checked for the ability to immunoprecipitate the homologous antigen. Immunodiffusion A 15 u1 sample of serum was tested by two-dimensional diffusion in agarose gels in 150 mM-NaCl/l0 mM-sodium phosphate buffer, pH 7.0, by the procedure of Ouchterlony & Nilsson (1978). RNA preparation, translation in vitro of the polyadenylated RNA, and immunoprecipitation and electrophoresis Total Sacch. cerevisiae and Schizosacch. pombe RNA was obtained from 100 ml of culture in mid-exponential phase (107 cells/ml) as described by Kaufer et al. (1985). Polyadenylated RNA was prepared by using oligo(dT)-cellulose as described by Clemens (1984). To 10 #u1 of rabbit reticulocyte lysate was added 1 ,l of polyadenylated RNA (1 ,ug/,ll) and 1 ,ul of [35S]methionine (10 Ci/,u1, 1150 Ci/mmol) and the mixture was incubated at 30 °C for 1 h. The reaction was stopped by adding I ,l of 10 % (w/v) sodium deoxycholate, and the mixture was diluted to 0.5 ml with 50 mM-Tris/HCl buffer, pH 7.5, containing 1 % Triton X- 100, 5 mM-EDTA, 50 mM-NaCl, 10 mM-methionine and 0.5 mg of BSA/ml. The immunoprecipitations were carried out in this buffer by adding 20 ,u of antiserum and 5 #1 of a solution of 200 mM-phenylmethanesulphonyl fluoride in ethanol. After incubation overnight at 4 'C, 50 ,l of 10% (w/v) inactivated Staph. aureus cells in 50 mM-Tris/HCl buffer, pH 7.4, containing 150 mM-NaCl, 5 mM-EDTA and 0.5 % Triton X-100 were added and the mixture was incubated for a further 30 min at 30 'C. Then the suspensions were spun down and the cells were washed with four 1 ml portions of 0.5 M-Tris/HCl buffer, pH 8.2, containing 0.1 % SDS, 7.5 % sucrose and 1 mg of BSA/ml and once with the same buffer without BSA. Eventually the cells were resuspended in electrophoresis sample buffer, boiled for 5 min and spun down, and the supernatant was loaded on a 10% polyacrylamide gel.
Peptide mapping Samples containing 2 ,g of deglycosylated invertase were treated with 10 ng of proteinase V8 from Staph. aureus for 20 min at room temperature in the stacking gel as described by Cleveland et al. (197-7). The -peptides were separated by SDS/ PAGE on a 14 %-polyacrylamide gel. 1990
Comparative analysis between two yeast invertases
699 IEF
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0
0
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Fig. 1. Chromatography of Schizosacch. pombe invertase on Bio-Gel A-5m Experimental conditions are described in the text. Each fraction contained 5 ml. 0, Protein; 0, invertase activity. The inset shows the electrophoretic analysis (in the absence of SDS) of the material containing the invertase activity, before (lane 1) and after (lanes 2 and 3) filtration through the Bio-Gel column, stained for protein with Coomasie Brilliant Blue (lanes 1 and 2) and for activity (lane 3), under the conditions described in the Materials and methods section.
RESULTS AND DISCUSSION Purification of invertase from Schizosacch. pombe As indicated in the Introduction, the goal of this work was to achieve the purification of invertase from Schizosacch. pombe in order to carry out its characterization. The initial approach used was to follow the method described for the purification of the same enzyme from Sacch. cerevisiae (Goldstein & Lampen, 1975); however, this method was found to be unsuitable for our purposes, since Schizosacch. pombe invertase was not retained by sulphoethyl-Sephadex columns and was eluted with the void volume of Sephadex G-200 columns, two of the purification steps of this method. Accordingly we had to develop a new procedure that would allow purification to apparent homogeneity of the invertase from Schizosacch. pombe cells fully de-repressed for the synthesis of the enzyme. This procedure is partly similar to that previously described for the purification of another Schizosacch. pombe exocellular glycoprotein enzyme, acid phosphatase (Dibenedetto & Cozzani, 1975), which in itself is an indication that both enzymes must share common physicochemical properties. The purification procedure described in the Materials and methods section for invertase takes advantage of three of the
Fig. 2. Two-dimensional electrophoresis of purified Schizosacch. pombe invertase Experimental conditions are described in the Materials and methods section. IEF represents isoelectric focusing (pH 3-10) (first dimension); SDS/PAGE represents polyacrylamide-gel (3-10 %) electrophoresis in the presence of 0.1 % SDS (second dimension). The gel was silver-stained.
most relevant properties of this enzyme: (1) its high solubility in concentrated salt solutions, probably due to its high carbohydrate content (see below), (2) its acidic isoelectric point (pl 3.25), which allows binding of the enzyme to anion-exchangers such as DEAESephadex A-50 at low pH values, and (3) its high molecular mass (Moreno et al., 1985; see below), which allows the separation of invertase from its major contaminant, maltase, after the DEAESephadex column by using a Bio-Gel A-Sm column (Fig. 1). The purity of the enzyme was assessed by two-dimensional gel electrophoresis; this revealed a single protein (Fig. 2). Table 1 summarizes the relevant purification data.
Molecular mass and subunit composition of invertase The molecular mass of the native purified invertase was determined by two different techniques: gel filtration in Bio-Gel A-5m and non-denaturing PAGE. In the first method, samples containing 7 units of invertase were gel-filtered through a Bio-Gel A-Sm column (1.25 cm x 100 cm) equilibrated with 0.1 M-Tris/HCl buffer, pH 7.5, containing 0.2 M-NaCl. An apparent molecular mass of 1200 kDa was extrapolated from a calibration curve obtained by gel-filtering, under the same conditions, samples of thyroglobulin (670 kDa), ferritin (440 kDa) and catalase (232 kDa). The elution volume of the invertase was not modified when a higher salt concentration (3 M-NaCl or 5 M-LiCI) or a detergent (1 % Cetrimide) was included both in the sample and in the elution buffer (results not shown). In the second method, samples of pure invertase containing 10 4ag of protein were electrophoresed on gel rods (6 mm x 10 cm) prepared with different concentrations of polyacrylamide, from
Table 1. Purification of Schizosacch. pombe invertase
For experimental details see the Materials and methods section.
Step Crude extract, pH 4.0 (NH4)2SO4 fractionation DEAE-Sephadex batch adsorption DEAE-Sephadex column chromatography Bio-Gel A-Sm column chromatography
Vol. 267
Total activity (units)
Total protein (mg)
Specific activity (units/mg)
196000 164000 74820 43720 7940
7630 526 146 36 5
26 313 512 1210 1590
S. Moreno, Y. Sanchez and L. Rodriguez
700 2.5 to 10%, under non-denaturing conditions along with the following molecular-mass markers: urease tetramer (480 kDa), urease dimer (240 kDa), BSA dimer (132 kDa), BSA monomer (66 kDa) and ovalbumin (45 kDa). A molecular mass for invertase of 1070 kDa was calculated by the procedure outlined in the Sigma Technical Bulletin no. MKR- 137 (results not shown). Subunit molecular mass was estimated by SDS/PAGE in 5 %polyacrylamide gel slabs in the presence of 8 M-urea both in the gel and in the sample buffer. A single smeared wide band was observed which was due to the glycoprotein nature of the enzyme, with a mean molecular mass of 205 kDa (results not shown). This suggests that the native enzyme could be constituted of five or six subunits of identical size.
Carbohydrate composition The glycoprotein nature of invertase and the presence of mannose and galactose in its carbohydrate moiety were inferred in a previous work (Moreno et al., 1985). In the present study the percentage of carbohydrate was determined as 67 % of the total mass of the protein. The individual sugars were identified by g.l.c. as described in. the Materials and methods section. Most of the carbohydrate was found to be mannose and galactose in a ratio of about 1:1. The enzyme was also shown to contain glucosamine, but in amounts too small to be quantified by this procedure. Glucosamine is probably involved in the linkage of the carbohydrate and the protein moieties of the enzyme molecule, through an N-glycosidic linkage, since the carbohydrate could be separated from the protein portion by treatment with endoglycosidase H under denaturing conditions (see the Materials and methods section). Fig. 3 shows that after this treatment the protein moved as a single and sharp band with a molecular mass close to 60 kDa. This value is in good agreement with the molecular mass calculated for invertase subunits, considering that their protein content is 33 % of the total weight. The sensitivity of invertase to endoglycosidase H is lost when the treatment is performed under non-denaturing conditions;
1
2
3
4
5
kDa
this may mean that the oligosaccharide present in the precursor of invertase undergoes some kind of post-translational processing during secretion to the cell wall, similar to that of animal cell glycoproteins (Hubbard & Ivatt, 1981; Dunphy & Rothman, 1985). Although we have been unable to detect the presence of other components of glycoproteins characteristic of higher eukaryotes, such as fucose or sialic acids, the presence ofgalactose in the sugar moiety of invertase and of acid phosphatase (Dibenedetto & Cozzani, 1975) may indicate that protein glycosylation in fission yeast takes place through a mechanism more similar to that found in animal cells, generating complextype or hybrid-type oligosaccharides in glycoproteins, than to that found in Sacch. cerevisiae, which generates glycoproteins with high-mannose-type asparagine-linked oligosaccharides (Kornfeld & Kornfeld, 1985). It would be interesting to express in Schizosacch. pombe higher-eukaryotic glycoproteins to check whether they are processed correctly and produce the right glycosylation pattern. Comparison of the protein fractions of the Schizosacch. pombe and Sacch. cerevisiae invertases After treatment with endoglycosidase H, both Schizosacch. pombe and Sacch. cerevisiae invertase gave rise to a polypeptide of similar molecular mass (Fig. 3). In order to establish the extent of the sequence similarity between the two proteins, we carried out a comparative analysis of both polypeptides. Purified enzymes were deglycosylated by treatment with endoglycosidase H. The protein fractions were purified by electrophoresis and electro-elution, as indicated in the Materials and methods section. Comparison of the amino acid compositions of the two enzymes (Table 2) revealed that they were similar except for the lysine contents. In order to investigate whether the two proteins shared common epitopes in the molecule, polyclonal antibodies were raised against both of them. The sera were tested by two-
Table 2. Comparison of the percentage amino acid compositions of Schizosacch. pombe and Sacch. cerevisiae invertases All values are averages from duplicate determinations after hydrolysis for 24 and 48 h, as described in the Materials and methods section. Abbreviations: N.D., not determined.
-205
Amino acid composition (%) -114 -97
-68
-60 -45
-40
Fig. 3. Effect of deglycosylation on the electrophoretic behaviour of the Schizosacch. pombe and Sacch. cerevisiae invertases Samples of purified invertases from Schizosacch. pombe (lanes 1 and 2) and Sacch. cerevisiae (lanes 3 and 4) were electrophoresed on an SDS/linear-gradient polyacrylamide (5-12 %) gel slab before (lanes 1 and 3) and after (lanes 2 and 4) treatment with endoglycosidase H. Experimental conditions are described in the text; the gel was stained with Coomassie Brilliant Blue. At the right-hand side of the gel are indicated the molecular masses (kDa) of the markers (lane 5), from top to bottom: myosin, 8l-galactosidase, phosphorylase b, catalase, ovalbumin and glyceraldehyde-3-phosphate dehydrogenase.
Amino acid
Schizosacch. pombe invertase
Sacch. cerevisiae invertase
Asx Thr Ser Pro Glx Gly Ala Val Cys Met Ile Leu Tyr Phe Lys Hys Trp
13.1 7.8 8.8 5.8 9.4 7.8 7.2 6.0 N.D. N.D. 5.4 7.6 5.6 4.4 2.0 1.9 N.D. 2.6
11.8 7.1 8.1 6.0 9.6 10.8 7.8 5.2 N.D. N.D. 5.5 6.4 3.8 5.8 6.9 2.4 N.D. 2.6
Arg
1990
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Comparative analysis between two yeast invertases (a).'
(b).
kDa
-.
2
3
4
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3
4
5
6
.. ......
60
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...
'.
.....
30-
20.1-
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5 _ 7 __
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Fig. 6. SDS/PAGE (14% polyacrylamide) of the invertases from Schizosacch. pombe (lanes 3 and 5) and Sacch. cerevisiae (lanes 4 and 6) before (lanes 3 and 4) and after (lanes 5 and 6) treatment with Staph. aureus proteinase V8 Lanes 1 and 2 are molecular-mass markers. The gel was silverstained.
..;_
205
g..:.^.: 114
97
-68
-
45
-36
Fig. 5. SDS/PAGE (10% polyacrylamide) of the peptides synthesized in vitro from Schizosacch. pombe polyadenylated RNA The translation products were immunoprecipitated with 20,ul of serum raised against invertase from Sacch. cerevisiae, in the presence of 0 ul (lane 1), 1 4u1 (lane 2), 2 ell (lane 3), 3 pi (lane 4) or 5 #1 (lane 5) of purified deglycosylated Sacch. cerevisiae invertase (1 mg/ml). Lanes 8 and 9 are the total products of the reaction in the absence and in the presence of mRNA respectively. The gel was dried and exposed to a Kodak XAR5 film for autoradiography.
dimensional immunodiffusion in agarose gels; in both cases immunoprecipitation bands were seen when the two invertases were used as antigen (results not shown). More interestingly, when the sera were tested for the ability to immunoprecipitate the heterologous antigen, immunoprecipitation bands on an immunodiffusion gel were seen (Fig. 4), indicating that each protein cross-reacts with antibodies raised against either of them. At this point, it was important to show that the two proteins Vol. 267
2
97-
Fig. 4. Cross-reaction between (a) Schizosacch. pombe invertase and serum raised against invertase from Sacch. cerevisiae and (b) Sacch. cerevisiae invertase and serum raised against invertase from Schizosacch. pombe Key: I = (1:1), 2 = (1:2), 3 = (1:4) etc. represent dilutions of the antigen.
1
1
cross-react because they share common epitopes within the protein fractions and not because of residual carbohydrate that could remain after the treatment with endoglycosidase H. To avoid this possibility, Schizosacch. pombe total RNA was prepared from cells grown under conditions in which synthesis of invertase was de-repressed. The RNA was translated in vitro in a rabbit reticulocyte lysate, in the presence of [35S]methionine. The products of the reaction were immunoprecipitated with antibodies raised against invertase from Sacch. cerevisiae. A protein of 60 kDa was immunoprecipitated (Fig. 5, lane 1). This band could be competed out when purified Sacch. cerevisiae invertase was added to the immunoprecipitation reaction (Fig. 5, lanes 2-5). A similar result was obtained when RNA from Sacch. cerevisiae was translated in vitro and the polypeptide mixture was immunoprecipitated with antibodies raised against invertase from Schizosacch. pombe (results not shown). Finally, peptide mapping of both proteins was carried out with the use of Staph. aureus proteinase V8. As shown in Fig. 6, the peptides generated were not identical, suggesting that the sequence similarity between the two enzymes might be limited to small regions of the proteins that are antigenically similar. To summarize, we have purified the Schizosacch. pombe invertase and shown that it is a high-molecular-mass glycoprotein composed of several identical subunits. The protein portion is very similar to that of the invertase from Sacch. cerevisiae, but the carbohydrate is different, resembling that of glycoproteins from higher eukaryotes. We thank Chris Norbury and Paul Nurse for their comments on the manuscript. This work was supported in part by Grant 0780/84 from the Comisi6n Asesora de Investigacion Cientifica y Tecnica and by predoctoral fellowships (to S.M. and Y.S.) from the Fondo de Investigaciones Sanitarias de la Seguridad Social.
REFERENCES Albersheim, P., Nevins, D. J. & English, P. D. (1967) Carbohydr. Res. 5, 340-345 An der Lan, B. & Chrambach, A. (1981) in Gel Electrophoresis of Proteins: A Practical Approach (Hames, B. D. & Rickwood, D., eds.), pp. 157-187, IRL Press, London and Washington Carlson, M. & Botstein, D. (1982) Cell 28, 145-154 Carlson, M., Osmond, B. C. & Botstein, D. (1981) Genetics 98, 25-40
S. Moreno, Y. Sanchez and L. Rodriguez
702 Clemens, M. J. (1984) in Transcription and Translation: A Practical Approach (Hames, B. D. & Higgins, S. J., eds.), pp. 211-230, IRL Press, Oxford Cleveland, D. W., Fischer, S. G., Kirschner, M. W. & Laemmli, U. K. (1977) J. Biol. Chem. 252, 1102-1106 Cohen, R. E. & Ballou, C. E. (1981) Encycl. Plant Physiol. New Ser. 13B, 441-458 Dibenedetto, G. & Cozzani, I. (1975) Biochemistry 14, 2847-2852 Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. (1956) Anal. Chem. 28, 350-356 Dunphy, W. G. & Rothman, J. E. (1985) Cell 42, 13-21 Gabriel, 0. &.Wang, S. F. (1969) Anal. Biochem. 27, 545-554 Gascon, S. & Ottolenghi, P. (1967) C. R. Trav. Lab. Carlsberg. 36, 85-93 Gascon, S. & Lampen, J. 0. (1968) J. Biol. Chem. 243, 1567-1572 Gascon, S., Neuman, N. P. & Lampen, J. 0. (1968) J. Biol. Chem. 243, 1573-1577 Goldstein, A. & Lampen, J. 0. (1975) Methods Enzymol. 42, 505-511 Hames, B. D. (1981) in Gel Electrophoresis of Proteins: A Practical Approach (Hames, B. D. & Rickwood, D., eds.), pp. 1-91, IRL Press, London and Washington
Hubbard, S. C. & Ivatt, R. J. (1981) Annu. Rev. Biochem. 50, 555-583 Huffaker, T. C. & Robbins, P. W. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7466-7470 Kaufer, N. F., Simanis, V. & Nurse, P. (1985) Nature (London) 318, 78-80 Kornfeld, R. & Kornfeld, S. (1985) Annu. Rev. Biochem. 54, 631-664 Laemmli, U. K. (1970) Nature (London) 227, 680-685 Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 Mitchison, J. M. & Creanor, J. (1969) J. Cell Sci 5, 373-391 Moreno, S., Ruiz, T., Sanchez, Y., Villanueva, J. R. & Rodriguez, L. (1985) Arch. Microbiol. 142, 370-374 Morrisey, J. H. (1981) Anal. Biochem. 117, 307-310 Neuman, N. P. & Lampen, J. 0. (1967) Biochemistry 6, 468-475 Ouchterlony, 0. & Nilsson, C. A. (1978) Handb. Exp. Immunol. 3rd Ed. 1, 19.1-19.44 Pollard, J. W. (1984) in Methods in Molecular Biology, vol. 1: Proteins (Walker, J. M., ed.), pp. 81-96, Humana Press, Clifton Tarentino, A. L., Plummer, T. H. & Maley, F. (1974) J. Biol. Chem. 249, 818-824
Received 19 September 1989/13 November 1989; accepted 24 November 1989
1990
